POS0456 AUTOPHAGY-RELATED RISK LOCI IN SLE AND THEIR ROLE IN NEUTROPHILS
Authors: A. Nagel, M. Radziszewski, B. Khatri, M. M. Wiley, A. M. Stolarczyk, M. L. Joachims, Q. Sun, K. Kim, S. C. Bae, B. Tsao, C. Lessard
Journal: Annals of the Rheumatic Diseases (2022)

Background Systemic lupus erythematous (SLE) is an autoimmune disease with ~150 established susceptibility risk loci. Genome wide-association (GWA) studies in SLE cases and controls of Korean ancestry identified the SLE risk locus ATG16L2-P2RY2, and rs11235604 as a SLE-associated missense variant (R220W) of Autophagy Related 16 Like 2 (ATG16L2) [1]. PRDM1-ATG5 is also an SLE risk locus in European populations that is implicated in autophagy. Autophagy plays a crucial role in neutrophil extracellular trap (NET) formation, degranulation, and limiting autoantigens in blood. Dysregulated autophagy has been implicated in SLE pathology and poor disease outcomes. The function of ATG16L2 is unknown, but evidence suggests it may function as a negative regulator of autophagosome formation [2]. Objectives To identify autophagy-related SLE risk variants shared across different ancestry populations and define the role of ATG16L2 in SLE and autophagy. Methods SLE case-control GWA scans from European (7568 cases; 1082 controls), African American (4336 cases; 935 controls), Hispanic (3752 cases; 1840 controls), and Korean (1173 cases; 4213 controls) populations were imputed and SNP associations tested. Meta-analysis was performed, then Bayesian statistics were used to define a credible SNP set. Bioinformatic analyses (RegulomeDB, promoter capture Hi-C, eQTLs, etc.) further prioritized SNPs based on predicted functionality. The functional significance of autophagy SLE risk genes, ATG16L1, ATG16L,2 and ATG5, were tested by CRISPR knockout (KO) in PLB-985 cell line. CRISPR-targeted single cell clones were screened for ATG16L1, ATG16L2 or ATG5 deletion using qPCR, NanoPore sequencing, and Western blotting. Changes in autophagy were assessed by Western blotting and confocal microscopy. Results Transracial fine-mapping of PRDM1-ATG5 locus identified two SNP associations shared across the credible sets in all populations: rs56886418 (p=1.38x10-5) located in the intron of PRDM1 and rs77791277 (p=1.38x10-5) that tagged a group of SNPs in strong linkage disequilibrium. Cross comparison of the credible SNP sets and bioinformatic analyses of shared SNPs identified rs533733 and rs9373843 as additional likely functional variants. Bioinformatic analyses prioritized rs56886418, an eQTL for ATG5 (p=0.05) and PRDM1 (p=4.75x10-7) in blood cells positioned in a topologically associated domain (TAD) that may interact with ATG5 and PRDM1 promoters in EBV-transformed B cells. SNP rs533733 is an eQTL for ATG5 in neutrophils (p=0.006) and is in a TAD 6.4kb 3’ of PRDM1 that interacts with the ATG5 promoter region where rs9373843 (eQTL of ATG5 in neutrophils (p=0.04)) is positioned. These data suggest that risk SNPs on the PRDM1-ATG5 locus may modulate ATG5 expression and autophagy in specific cell types by modulating the local chromatin regulatory network. To assess the roles of ATG5, ATG16L1 and ATG16L2 in autophagy, PMA/I-induced hallmarks of autophagy, LC3-I and LC3-II conversion and p62 protein aggregation, were assessed in homozygous and heterozygous ATG5, ATG16L1, or ATG16L2 CRISPR KO PLB-985 cells by Western blotting and confocal microscopy. Loss of ATG5 or ATG16L1 impaired PMA/I-induced autophagosome formation in myeloid-like and differentiated neutrophil-like PLB-985 cells. In contrast, loss of ATG16L2 elevated the conversion of LC3-I to LC3-II and p62 protein aggregation in both cell types, suggesting that ATG16L2 may inhibit autophagy. Conclusion Functional characterization of SNPs on the PRDM1-ATG5 and ATG16L2-P2RY2 loci, and the functional characterization of ATG16L2 in myeloid and neutrophil cell lines, provide new insights into the mechanisms that regulate autophagy in health and disease. Ongoing studies will focus on in vitro validation of predicted functional SNPs and will introduce ATG16L2 rs11235604 risk variant in PLB-985 cells to assess its importance in autophagy. References [1]Lessard CJ, et al. Arthritis Rheumatol. 2016; 68(5):1197-1209. [2]Wible DJ, et al. Cell Discov. 2019; 5:42. Disclosure of Interests None declared.